JP2014510146A - BPI and the like as radiation relaxation agents and radiation protection agents - Google Patents

Abstract

Described herein are radiation exposure (incidental / unintentional or intentional (such as treatment)), chemoradiotherapy, disease, toxins, or drugs or organisms in a subject (individual). A method of alleviating tissue injury resulting from mediated therapy.

Description

Research Assisted by the Federal Government This invention was made with government support under the Defense Advanced Research Projects Agency (DARPA) Grant HR001-08-0011 and Center for Medical Countermeasures Research Pilot Grant 5U19A1067751. Accordingly, the government has certain rights in the invention.

Background of the Invention Human exposure to radiation can cause serious harm, even death. The exposure can be accidental, for example from a radiation leak from a nuclear power plant. The exposure can also be intentional, eg resulting from acts of terrorism. The most common situation of radiation exposure arises from medical interventions such as cancer treatment. Radiation in this context can be local or systemic. When applied topically, radiation can nevertheless cause unwanted damage to healthy tissue in the path of radiation. When applied systemically (ie whole body irradiation), low doses can result in bone marrow damage and toxicity to the gastrointestinal tract. High doses of whole body irradiation can result in permanent bone marrow damage, intestinal and lung toxicity, and sometimes death. There is a need for effective treatments that protect healthy tissue and mitigate the acute and chronic effects of exposure to ionizing radiation.

SUMMARY OF THE INVENTION Described herein is that in a subject (individual), exposure to radiation (accidental / unintentional or intentional (such as treatment)), chemoradiotherapy or disease resulting from a disease. It is a way to alleviate injury. The method includes subjecting an individual (individual) referred to as a subject in need thereof to a bacterial / permeability increasing protein (BPI), a BPI analog of (at least 1, 1 or 2 or more), Or both BPI and BPI congeners are administered in an amount sufficient to reduce (partially or completely) the effects of exposure, thereby mitigating tissue damage resulting from radiation exposure in the subject Including that. In certain embodiments, the radiation exposure results from accidental exposure to radiation, such as occurs in a nuclear plant failure event, or from intentional exposure to radiation, such as therapeutic radiation, chemotherapy or radiation therapy. The tissue injury can be, for example, injury to hematopoietic tissue (eg, bone marrow) or injury to the gastrointestinal (GI) tract. In certain embodiments, the tissue injury is hematopoietic toxicity. BPI congeners used in the method include, but are not limited to, rBPI ₂₁ , rBPI ₂₃ , rBPI ₅₀ , rBPI (10-193) ala ^132, and N-terminal fragments of BPI having an approximate molecular weight of about 20 kD to about 25 kD. It is done.

  In certain embodiments, the BPI and / or the like are administered between 1 day prior to exposure of the subject to radiation and 2 days (48 hours) after exposure of the subject to radiation. BPI and / or the like can be administered orally, intravenously, or subcutaneously.

  In some embodiments, a method of alleviating tissue injury resulting from exposure to radiation (accidental / unintentional or intentional (such as treatment)), chemoradiotherapy or disease in a subject further comprises Administration of (at least 1, 1 or 2 or more) antibiotics to a subject in need. The antibiotic may be, for example, a quinolone antibiotic such as an antibiotic selected from the group consisting of moxifloxacin, ciprofloxacin, levofloxacin, garenoxacin and delafloxacin.

In another aspect, the method is mediated in a subject (individual) by exposure to radiation (accidental / unintentional or intentional (such as treatment)), chemoradiotherapy, disease, toxin or drug or organism. A method to alleviate hematopoietic toxicity resulting from treatment. The method can be applied to a subject (individual) referred to as a subject in need of bactericidal / permeability enhancing protein (BPI), (at least one, one or more) BPI congeners, or BPI and BPI congeners Administration of both in a quantity sufficient to alleviate (partially or completely) the hematopoietic toxicity of the subject. BPI congeners used in the method include, but are not limited to, rBPI ₂₁ , rBPI ₂₃ , rBPI ₅₀ , rBPI (10-193) ala ¹³² , and an N-terminal fragment of BPI having an approximate molecular weight of about 20 kD to about 25 kD. Can be mentioned.

  In certain embodiments, the BPI and / or the like are administered between 1 day prior to exposure of the subject to radiation and 2 days (48 hours) after exposure of the subject to radiation. BPI and / or the like can be administered orally, intravenously, or subcutaneously.

  In some embodiments, in a subject (individual), from exposure to radiation (accidental / unintentional or intentional (such as treatment)), chemoradiotherapy, disease, toxin or drug or biologically mediated therapy The method of alleviating resulting hematopoietic toxicity further comprises administering an antibiotic (at least one, one or more) to the subject (a subject in need thereof). The antibiotic may be, for example, a quinolone antibiotic such as an antibiotic selected from the group consisting of moxifloxacin, ciprofloxacin, levofloxacin, garenoxacin and delafloxacin.

A further aspect is a method for bone marrow recovery in a subject (individual), said method comprising subjecting a subject (individual) referred to as a subject in need thereof to bactericidal / permeability enhancing protein (BPI), ( Administering at least one, one or more BPI congeners, or both BPI and BPI congeners, in an amount sufficient for bone marrow recovery in a subject. BPI congeners used in the method include, but are not limited to, rBPI ₂₁ , rBPI ₂₃ , rBPI ₅₀ , rBPI (10-193) ala ^132, and N-terminal fragments of BPI having an approximate molecular weight of about 20 kD to about 25 kD. It is done.

  In certain embodiments, the BPI and / or the like are administered between 1 day prior to exposure of the subject to radiation and 2 days (48 hours) after exposure of the subject to radiation. BPI and / or the like can be administered orally, intravenously, or subcutaneously.

  In some embodiments, the subject has one or more hematopoietic cell types or lineage deficiencies. For example, the subject may be lymphopenia, myelopenia, leukopenia, neutropenia, erythropenia, megakaryopenia, platelet deficiency, monocyte deficiency, lymph It may have hematopoietic defects such as sphere deficiency, red blood cell deficiency, neutrophil deficiency, T cell deficiency, granulocyte deficiency, and / or dendritic cell deficiency. Depletion of one or more hematopoietic cell types or lineages can result from exposure to radiation, chemoradiotherapy, radiation therapy diseases, toxins, or drugs or biologically mediated therapy.

  In some embodiments, exposure (incidental / unintentional or intentional (such as treatment)), chemoradiotherapy, disease, toxin, or drug or organism mediated treatment in a subject (individual). The method of bone marrow recovery that may result from further comprises administering an antibiotic (at least one, one or more) to the subject (subject in need thereof). The antibiotic may be, for example, a quinolone antibiotic such as an antibiotic selected from the group consisting of moxifloxacin, ciprofloxacin, levofloxacin, garenoxacin and delafloxacin.

A further method is a method for stimulating hematopoiesis in a subject (individual), said method comprising subjecting a subject (individual) referred to as a subject in need thereof to bactericidal / permeability enhancing protein (BPI), Administering (at least one, one or more) BPI congeners, or both BPI and BPI congeners, in an amount sufficient to stimulate hematopoiesis in a subject. BPI congeners used in the method include, but are not limited to, rBPI ₂₁ , rBPI ₂₃ , rBPI ₅₀ , rBPI (10-193) ala ^132, and N-terminal fragments of BPI having an approximate molecular weight of about 20 kD to about 25 kD. It is done.

In some embodiments, the subject has one or more hematopoietic cell types or lineage deficiencies. Hematopoietic deficiency means, for example, lymphopenia, myelocytopenia, leukopenia, neutropenia, erythrocytopenia, megakarycytopenia, platelet deficiency, monocyte deficiency, lymphocyte There may be a deficiency, a red blood cell deficiency, a neutrophil deficiency, a T cell deficiency, a granulocyte deficiency, and / or a dendritic cell deficiency. Depletion of one or more hematopoietic cell types or lineages results, for example, from radiation exposure, chemoradiotherapy, radiation therapy, disease, toxins, or treatments mediated by drugs or organisms. BPI congeners used in the method include, but are not limited to, rBPI ₂₁ , rBPI ₂₃ , rBPI _50, rBPI (10-193) ala ¹³² and N-terminal fragments of BPI having an approximate molecular weight of about 20 kD to about 25 kD. It is done.

  In certain embodiments, the BPI and / or the like are administered between 1 day prior to exposure of the subject to radiation and 2 days (48 hours) after exposure of the subject to radiation. BPI and / or the like can be administered orally, intravenously, or subcutaneously.

  In certain embodiments, the method for stimulating hematopoiesis in a subject (individual) further comprises administering an antibiotic (at least one, one or more) to the subject (subject in need thereof). The antibiotic may be, for example, a quinolone antibiotic such as an antibiotic selected from the group consisting of moxifloxacin, ciprofloxacin, levofloxacin, garenoxacin and delafloxacin.

  As described herein, in certain embodiments, the method comprises: (a) bactericidal / permeability enhancing protein (BPI), (at least one, one or more) BPI analogs or BPI and BPI analogs And (b) administering (at least one, one or more) antibiotics to a subject (individual) in need thereof. In these embodiments, the bactericidal / permeability enhancing protein (BPI), BPI analog, or both BPI and BPI analogs, and the antibiotic can be administered simultaneously (together) or sequentially. When bactericidal / permeability enhancing protein (BPI), BPI congeners or both BPI and BPI congeners and antibiotics are administered simultaneously, they are either in one composition or in separate compositions. Can be administered. When bactericidal / permeability enhancing protein (BPI), BPI congeners or both BPI and BPI congeners and antibiotics are administered sequentially, they may be administered in any order, They need to be administered at a time close enough so that they have the desired palliative effect.

  The subject (individual) is an animal, typically a mammal. In one aspect, the subject is a dog, cat, horse, sheep, goat, cow or rodent. In important embodiments, the subject is a human. In any of the foregoing embodiments, the subject does not otherwise require treatment with BPI and / or the like. In some such embodiments, the subject does not have an infectious disease.

  These and other aspects of the invention, as well as various advantages and uses, will become apparent upon reference to the detailed description. As will be appreciated, each aspect of the invention may encompass a variety of embodiments.

FIG. Changes in circulating levels of neutrophils (ANC) and platelets (PLT), mCD14 and TLR4 levels on the monocyte surface, plasma levels of endotoxin, BPI and IL-6, and the occurrence of fever were observed in human bone marrow disruption HSCT. Observed later. (A) Severe neutropenia (n = 46) and thrombocytopenia occurred (n = 48, lowest point D7). The data represents the geometric mean + SEM of logarithmic transformation values expressed in the original units. (B) In 18 patients, plasma endotoxin was assessed by endotoxin activity assay (EAA) and baseline (B; n = 17) as well as D0 (n = 17), D7 (n = 10) after bone marrow destruction, D14 (n = 15), D21 (n = 15) and D28 (n = 3) were reported in EAA units. A horizontal dashed line (in 0.4EA units) indicates the lower limit of detection (LLD). Plasma BPI concentrations in pg / mL were determined by ELISA using B (n = 48), D0 (n = 46), D7 (n = 48), D14 (n = 48), D21 (n = 47) and D28 (n = 33). The dotted line indicates the LLD (<100 pg / ml) for the BPI ELISA. Samples below LLD were assigned a value of 50% of LLD. (C) Measurement of surface expression of monocyte mCD14 and TLR4 by flow cytometry reveals the lowest point for mCD14 at D0 (n = 10) and the peak of simultaneous TLR4 expression at D0 (n = 9) It became. Data represent the geometric mean + SEM of logarithmic transformation values expressed in original units of mean fluorescence intensity (mCD14) or binding index (TLR4). (D) Plasma IL-6 (n = 37) and fever generation (n = 48) both had a peak at D7. IL-6 data represents the geometric mean + SEM of logarithmic transformation values expressed in original units.

FIG. BALB / c mice show BM loss and death at 7 Gy. (A) Mortality at D30 after TBI with different doses of 6 Gy (n = 10), 6.5 Gy (n = 20) and 7 Gy (n = 20) (p <0.001, Mantel-Cox log rank) ). (B) Rapid onset of mucosal damage was demonstrated by the peak of colonic epithelial cell death at D3 (n = 5) after TBI, which was expressed normalized to the level before TBI (D0 = 100%) Occurs simultaneously with the lowest point in plasma citrulline levels (n = 7). Data represent mean ± SEM. * P <0.05 is analyzed by 1 sample t-test compared to D0; ** p <0.001 is by Mann-Whitney. (C) Representative H & E stained femoral section shows BM disappearance at D3 after 7 Gy TBI (4 × magnification). (D) 0 Gy (normal control, n = 3 / time point), 6.5 Gy (n = 8 / time point), and 7 Gy (n = 8 on day 3 and day 10, higher mortality at D15) BM MNC after n = 6). Data are mean ± SD of individual counts. After 7 Gy, there was less BM MNC for 6.5 Gy (D3: p = 0.05, D10: p = 0.0002, D15: p = 0.02). Flow cytometric analysis of LK (E) and LSK cells (F) in the same mouse BM showed that 7 Gy resulted in an extended decrease in the number of precursors and HSCs. By D15, 6.5 mice had higher LK and LSK cell numbers than 7 Gy mice (p = 0.01 for both LK and LSK). Each symbol represents the absolute number of LK or LSK in the BM from one limb of an individual animal. Median / group is indicated by a horizontal bar. Hematology data was analyzed by Mann-Whitney.

FIG. RBPI ₂₁ combined with ENR increases the survival rate of BALB / c mice after 7 Gy TBI. (A) Survival rate of mice irradiated with 7 Gy and given ENR plus rBPI ₂₁ or VEH, ENR alone for 30 consecutive days 24 hours after irradiation, or untreated (labeled 7 Gy). In a combined analysis of 3 replicate experiments, rBPI ₂₁ / ENR-treated mice outperformed the other groups (p <0.0001 by Mantel-Cox log rank, n = 70 mice / arm). The survival rate of the rBPI ₂₁ / ENR group also exceeded that of VEH / ENR, ENR and 7 Gy (P <0.0001, 0.008 and <0.0001, respectively, according to the pair-wise Mantel-Cox log rank). (B) 7 hours after irradiation of 7 Gy of given rBPI ₂₁ or VEH irradiation (continued for 14 or 30 days), plus ENR (continued for 30 days) or untreated (labeled 7 Gy) Survival rate. Survival was not affected by the duration of rBPI ₂₁ treatment. Data were analyzed by pairwise Mantel-Cox log rank (n = 20 mice / group).

FIG. rBPI ₂₁ / ENR accelerates the recovery of hematopoiesis after dysplasia induced by TBI. The BALB / c BM MNC count (from one hind limb) and histopathology (from the contralateral hind limb) were evaluated 10, 15, and 19 days after the various treatments. (A) Data shown for untreated, age-matched controls (normal) or (b) 7 Gy irradiated mice. Other mice received both 7 Gy TBI and subsequent treatment started 24 hours after irradiation: (C) ENR, (D) VEH / ENR or (E) rBPI ₂₁ / ENR. Left panel: Each graph shows a count of BM MNC washed out from the hind limbs of 8 mice / group (mean ± SD), except that (B) high mortality experienced by mice given only 7 Gy Except where rate (median survival is 12-15 days) yields n = 2-8 / time points. According to Mann-Whitney, the combination of rBPI ₂₁ / ENR is compared to 7 Gy, ENR and VEH / ENR as D10 (p = 0.0003, 0.001 and <0.0001, respectively), D15 (respectively p = 0.0007, p = 0.001 and p = 0.001) and D19 (p = 0.006, p <0.0001 and p <0.0001, respectively) improve BM cellularity It became the result. Data was collected from two replicate studies. Similar results were obtained from both studies. Right panel: Representative D19 femoral H & E stained sections of animals receiving the indicated treatment show a close correlation between BM MNC counts and BM histology.

FIG. rBPI ₂₁ / ENR treatment results in restoration of BM to a normal level of cellularity by D30 after irradiation. BM histopathology (one hind limb) and MNC count (contralateral hind limb) were evaluated in mice that survived to D30. Representative thigh histology is shown for (A) untreated, age matched controls (normal) or (B) 7 Gy irradiated mice. Other mice received both 7 Gy TBI and subsequent treatment started 24 hours after irradiation: (C) ENR, (D) VEH / ENR or (E) rBPI ₂₁ / ENR. In addition to the histology, the count of the corresponding BM MNC washed away from the hind limbs of individual mice was determined (F). Bars represent mean ± SD for n = 4, 3, 12, 16, and 7 mice / group, respectively. The initial high mortality of mice treated with 7Gy alone and VEH / ENR limited the size of these cohorts. Only rBPI ₂₁ / ENR treatment resulted in BM MNC counts that were statistically distinguishable from 0 Gy. The rBPI ₂₁ / ENR MNC count was also different from the count in 7 Gy, ENR and VEH / ENR (p = 0.01, p = 0.0002, p = 0.001, respectively). Data from two replicate studies are shown. Similar results were obtained in all studies. Data was analyzed by Mann-Whitney.

FIG. rBPI ₂₁ / ENR treatment is associated with faster proliferation of early hematopoietic cells after 7 Gy TBI. Using flow cytometry, an unmatched untreated control (0 Gy) or 7 Gy was administered, and 24 hours later, no treatment (7 Gy), ENR, rBPI ₂₁ / ENR or VEH / ENR treatment was initiated LK (left panel) and LSK (right panel) cells contained in mouse BM MNCs were quantified. Results from D10 (upper panel), D19 (middle panel) and D30 (lower panel) are shown. The box graph represents the range of LK or LSK phenotype cells in the BM, 25th and 75th percentiles, and median number from one hind limb of each animal in each treatment group. N = 4 for 0 Gy control at all time points. N = 8 mice / treatment at D10. N = 6-8 mice / treatment at D19. Higher inequality in survival up to D30 resulted in n = 3 (7 Gy), 12 (ENR), 16 (rBPI21 / ENR) and 7 (VEH / ENR) mice / group. Compared to 7Gy, ENR or VEH / ENR, rBPI ₂₁ / ENR treatment was associated with a higher number of both LK and LSK cells at an early time point (p = 0.004 for all comparisons in D10, In D19, p = 0.004, 0.0003, and 0.0001, respectively. The LK and LSK contents of D30 for all groups including controls were equal. Data from two replicate experiments are shown. Similar results were obtained in all studies. Data was analyzed by Mann-Whitney.

FIG. 7Gy irradiation of BALB / c mice is associated with subsequent endotoxemia. For endotoxin assays with LAL, blood samples were obtained on the indicated days and expressed as mean ± SEM. Endotoxin has been present since D3. N = 9 mice / time point at days 0, 3, 12; n = 6 at D6 and n = 8 at D9. 7 Gy-only treatment mortality rate is sufficient for analysis beyond D12 Hindered.

FIG. Injection site injury and inflammation result from BID injection of rBPI ₂₁ or VEH. During these radiation mitigation studies, some 7 Gy irradiated BALB / c mice received only oral ENR. Other 7 Gy irradiated mice were treated with 250 μl of rBPI ₂₁ or its formulation buffer (VEH and VEH) using ENR and a sterile single use insulin needle with a fixed 28.5 G needle twice daily. Received injection). The injection was started 24 hours after radiation and continued until day 30. On day 15 (B) or day 19 (A, C), mice were humanely euthanized and the underside of ventral skin was exposed for photographic examination of local tissue injury. Images were acquired using a Nikon D90 digital camera.

FIG. Three blood cell hematopoiesis in cellular BM of rBPI ₂₁ / ENR treated mice. BALB / c mice were irradiated with 7GY, and rBPI ₂₁ / ENR was started 24 hours later. Mice were euthanized 19 days after irradiation. A low magnification image of a femoral H & E stained coronal section in rBPI ₂₁ / ENR treated mice is shown in FIG. These images show higher magnification images at (A) 20 × and (B) 40 ×. BM showed trihemecellular hematopoiesis without dysplasia, relative bone marrow hyperplasia, and strong megakaryocyte recovery.

FIG. Gating strategy to determine LK and LSK cells in BM from BALB / c mice by FACS. To exclude small debris, a gate is drawn on the dot plot of BM cell FSC vs. SSC. Committed lineage cells are determined in FL-4 channels (APC positive) vs. SSC and gated to bifurcate these from negative (negative to low APC fluorescence) cells for lineage marker expression. Drew. The gate was confirmed through the use of a matching isotype control cocktail (also conjugated to APC). Lineage negative cells are visualized on a Sca-1PE x c-kit-PerCP5.5 double fluorescent dot plot to show Lin ⁻ Sca-1 ⁻ c-kit ⁺ (LK precursor cells) and Lin ^{− in} mouse BM. The content of Sca-1 ⁺ c-kit ⁺ (LSK stem cells) was evaluated. The histogram shown is from an analysis of normal mice.

FIG. Limited pilot clinical trials of rBPI ₂₁ infusion in patients undergoing allogeneic HSCT support tolerance after bone marrow destruction treatment. Four of six patients were in the first cohort of a phase I-II pilot trial of rBPI ₂₁ administration in a multi-institutional, radiation-based treatment of myeloablative HSCT approved by the IRB Registered. All had hematological malignancies, were 50-65 years old (median 55), and signed a consent form. Subjects received cyclophosphamide and 1360 (n = 3) or 1400 (n = 1) split TBI for pretreatment of bone marrow destruction. (A) All patients received 4 mg / kg bolus intravenous rBPI ₂₁ on day 1, followed by continuous intravenous infusion over 6 hours at 6 mg / kg / day. The dose and duration of continuous infusion was increased according to the indicated schedule, but the study was interrupted by a supporter (XOMA (US) LLC) when the drug study lots became obsolete. (B) Shows serious adverse effects during the introduction of HSCT.

FIG. The effects of ENR added to 14 and 30 days of rBPI ₂₁ on bone marrow mononuclear cells, LK and LK cells are comparable. BALB / c mice were irradiated with 7 Gy, and treatment was started 24 hours later. Some mice were dosed twice daily with subcutaneous rBPI ₂₁ in combination with ENR up to D15, at which point the rest was called a group called rBPI ₂₁ (14) / ENR (rBPI ₂₁ was discontinued) ENR lasts up to D30), and were equally divided into another group rBPI21 (30) / ENR (to sustain both rBPI ₂₁ and ENR to D30). The VEH / ENR group was treated as described above. rBPI ₂₁ (14) / ENR and rBPI ₂₁ (30) / ENR are at the same level at the same level of bone marrow mononuclear cells (Panel A, D), LK (Panel B, E) and LSK (Panel C, F ). Quantification was performed as per method column. Bar graph + SD represents bone marrow mononuclear cells and box graph represents range of LK or LSK phenotype cells in bone marrow from one hind limb of each animal in each treatment group, 25th and 75th percentiles As well as the median number. For the rBPI ₂₁ (14) / ENR and rBPI ₂₁ (30) / ENR groups, D15: n = 4 / group, D18: n = 5-6 / group and D30: 8-10 / group. Due to mortality, there were n = 1 (7 Gy) and n = 2 (VEH / ENR) at D18 and no survivors in these groups at D30. Values obtained from two normal animals are expressed as 0 Gy values. Data from one study are shown.

FIG. Peripheral blood counts are comparable after ENR treatment in addition to 14 or 30 days of rBPI ₂₁ . In the peripheral blood of BALB / c mice treated with rBPI ₂₁ for 14 days (rBPI ₂₁ (14) / ENR-red circle), treated with rBPI _{21 for} 30 days (rBPI ₂₁ (30) / ENR-gray circle) In comparison, comparable levels of white blood cells (WBC), neutrophils, monocytes, platelets and hemoglobin were measured. Treatment started 24 hours after 7 Gy irradiation. Peripheral blood counts were obtained as described in the method. All mice were dosed twice daily with subcutaneous rBPI ₂₁ in combination with ENR up to D15, at which time blood was collected from 4 mice for peripheral blood cell analysis. The rest is a group called rBPI ₂₁ (14) / ENR (rBPI ₂₁ discontinues but ENR persists until D30), and another group rBPI ₂₁ (30) / ENR (both rBPI ₂₁ and ENR up to D30) To last) Results show the mean + standard deviation of peripheral blood counts measured at D18 (n = 5-6 / group) and D30 (8-10 / group). Values obtained from two normal animals are expressed as D0 values. Data from one study are shown.

FIG. (AF) Granulocyte stimulating factor (G-CSF) levels increase in response to rBPI ₂₁ treatment in irradiated and unirradiated mice. FIG. (AC) Mouse keratinocyte chemotactic factor (mouse KC) levels increase in unirradiated mice in response to rBPI ₂₁ treatment. Stimulation of mouse KC by rBPI ₂₁ treatment is enhanced by prior irradiation. FIG. Monocyte chemotactic protein-1 (MCP-1), also known as chemokine (CC motif) ligand 2 (CCL2) levels are increased by rBPI ₂₁ treatment.

DETAILED DESCRIPTION OF THE INVENTION The present invention, in one aspect, provides antibacterial / permeability enhancing protein (BPI) and / or the like, alone or in combination with antibiotics, from exposure to radiation, chemotherapy or disease. It relates to the surprising discovery that the resulting tissue injury can be alleviated. As described herein, BPI and / or the like, alone or in combination with antibiotics, in subjects (individuals) exposed to accidental or incidental radiation, And / or can be used to alleviate hematopoietic toxicity, stimulate hematological function, and assist bone marrow recovery in subjects with severe bone marrow destruction with bone marrow cell depletion / failure .

  As used herein, a subject in need of treatment with BPI and / or the like is a subject that has a (partial or complete) reduction in bone marrow function. In some embodiments, the subject has insufficient hematopoiesis in one or more blood cell types or blood cell lineages. In some embodiments, the subject has been exposed to radiation, chemoradiotherapy or a toxin, or a disease that results in (partial or complete) reduction in bone marrow function in the subject, or a drug or organism-mediated hematopoietic injury Have Examples of diseases that cause bone marrow function (partial or complete) include, but are not limited to, acute and chronic inflammation, infection, aplastic anemia, Fancony anemia, Bloom syndrome, reticular dysgenesis ), Costman syndrome, congenital benign neutropenia, neonatal sepsis, myelodysplastic syndrome, diamond blackfan anemia, and congenital or acquired bone marrow failure syndrome. The subject does not otherwise require treatment with BPI and / or the like. In some such embodiments, the subject does not have an infectious disease.

  In some embodiments, the subject is exposed to a level of radiation sufficient to cause undesired tissue injury and / or hematopoietic toxicity. In some embodiments, the subject has been exposed to sufficient radiation to cause tissue injury and / or hematopoietic toxicity prior to treatment with BPI and / or the like. In some embodiments, the subject is not exposed to radiation and predicts future radiation exposure prior to exposure to sufficient radiation to cause tissue injury and / or hematopoietic toxicity, and BPI and Receive treatment with / or the like. In some embodiments, the subject is exposed to radiation during treatment with BPI and / or the like. Radiation exposure includes, but is not limited to, exposure resulting from medical radiation therapy such as accidental exposure, nuclear attack, local treatment, and low and high doses of total body irradiation. In some embodiments, the subject has cancer and is experiencing, is currently undergoing, or is undergoing radiation therapy, chemoradiotherapy and / or chemotherapy.

  In accordance with some aspects of the present invention, a method is provided for mitigating tissue injury induced by any type of radiation. Exposure to radiation is toxic at low doses and life threatening at high doses. The tissues most vulnerable to radiation-induced damage include the hematopoietic system and the gastrointestinal tract (GI). Moderate radiation can cause a rapid decrease in blood cell counts, including loss of circulating lymphocytes and a decrease in mitotically active hematopoietic progenitor cells. A decrease in blood count is associated with, among other things, an increased risk of infection and the development of cancer. Higher doses of radiation can result in more severe and often permanent bone marrow damage resulting from the loss of bone marrow stem cell population. Thus, tissue injury can be, for example, a decrease in blood cell count, loss of bone marrow stem cell population, or hematopoietic toxicity.

  According to some aspects of the invention, a method for mitigating hematopoietic toxicity is provided. The method includes administering BPI and / or the like, alone or in combination with antibiotics. The term “hematopoietic toxicity” refers to toxicity that substantially results from exposure to radiation and that adversely affects the hematopoietic system of an individual (subject). Alternatively, hematopoietic toxicity may result from exposure of a subject to toxins, or a disease or genetic predisposition to hematopoietic injury. This adverse effect may be manifested extensively in subjects in the form of a change in the level of many hematopoietic cell types as radiation exposure, chemotherapy, toxins or disease (different from what is considered normal) Alternatively, the adverse effect is more specific in the subject in that it differs from the level at which only one or a few hematopoietic cell types are considered normal as a result of exposure to radiation, chemotherapy, toxins or disease. It may be manifested.

  BPI and / or the like and antibiotics are administered to alleviate tissue injury such as hematopoietic toxicity. As used herein, the term “alleviate” means a reduction in the degree of tissue damage induced by disease, chemotherapy, toxins or radiation (injury occurs in the absence of BPI / conservative treatment). It is lighter than the one that would be. Thus, a reduction in the extent of tissue damage induced by disease, chemotherapy, toxins or radiation can be assessed by improving tissue health in the subject being treated. Improving the tissue health of the subject being treated improves the tissue health in the subject being treated as a control subject (a subject receiving the same amount of radiation exposure as the subject being treated but not receiving BPI therapy). Can be determined by testing for tissue health. Tissue health can be measured by any of a variety of methods known to those skilled in the art, including direct and indirect measurements. A direct measurement is such as measuring cell counts. In some embodiments, the measured tissue injury may be tissue necrosis and / or a decrease in blood cell count. In some embodiments, improved tissue health comprises hematocrit, leukocyte count, incorporation of tritium-labeled thymidine into bone marrow DNA, spleen weight, burst forming units from bone marrow obtained from spleen or humerus or thigh. Endpoints such as the number of red blood cells or colony forming units (series forming red blood cells, granulocytes, macrophages and megakaryocytes) or enumeration of circulating hematopoietic stem cells or other primitive hematopoietic cells in the peripheral circulation And can be measured by evaluating the function of the hematopoietic system.

  According to some aspects of the invention, a method for bone marrow recovery is provided. The method includes administering BPI and / or the like, alone or in combination with antibiotics. “Bone marrow recovery” means that bone marrow damaged by radiation, chemotherapy, disease or toxin is restored to its normal or near normal state (function) or measurable improvement of bone marrow function Means the process of obtaining Bone marrow function is the process by which various blood cell types or lineages are produced from hematopoietic (blood) stem cells. Endpoints that can be used to measure bone marrow recovery include, but are not limited to, hematocrit, leukocyte counts, incorporation of tritium-labeled thymidine into bone marrow DNA, spleen weight, bone marrow obtained from spleen or humerus or thigh The number of erythrocytes in burst forming units or the number of colony forming units (series forming red blood cells, granulocytes, macrophages and megakaryocytes), or the count of circulating hematopoietic stem cells or other primitive hematopoietic cells in the peripheral circulation It is done. In some embodiments, the subject has been exposed to radiation or toxins or has a disease or drug- or organism-mediated hematopoietic injury that results in (partially or completely) reduced bone marrow function in the subject. Examples of diseases that cause bone marrow function (partial or complete) include, but are not limited to, acute and chronic inflammation, infection, aplastic anemia, Fancony syndrome, Bloom syndrome, reticulodysplasia, costman Syndromes, congenital benign neutropenia, neonatal sepsis, myelodysplastic syndrome, diamond blackfan anemia, and congenital or acquired bone marrow failure syndromes.

  According to some aspects of the invention, a method is provided for stimulating hematopoiesis. The method includes administering BPI and / or the like, alone or in combination with antibiotics. “Stimulation of hematopoiesis” generally refers to an increase in one or more hematopoietic cell types or lineages, particularly if the subject has a deficiency in one or more hematopoietic cell types or lineages. It relates to stimulation or enhancement of two or more hematopoietic cell types or lineages. Depletion of one or more hematopoietic cell types or lineages can be caused by radiation or toxin exposure, disease, drugs or organism-mediated hematopoietic injury. Examples of diseases that cause a deficiency in one or more hematopoietic cell types or lineages include, but are not limited to, acute and chronic inflammation, infection, aplastic anemia, Fancony syndrome, Bloom syndrome, reticulodysplasia, Costman syndrome, congenital benign neutropenia, neonatal sepsis, myelodysplastic syndrome, diamond blackfan anemia, and congenital or acquired bone marrow failure syndrome. Hematopoietic defects include lymphopenia, leukopenia, neutropenia, erythrocytopenia, megakarycytopenia, platelet deficiency, monocyte deficiency, lymphocyte deficiency, erythrocyte deficiency, neutrophil Deficiency, T cell deficiency, or B cell, especially granulocyte deficiency, and / or dendritic cell deficiency.

The compounds useful in the methods of the present invention are BPI, biologically active fragments, analogs, variants and / or the like. BPI, a 50-55 kDa cationic antibacterial protein originally found in the azurophilic granules of human polymorphonuclear neutrophils, is an endotoxin associated with a variety of bacteria and a cell-free form of endotoxin. Has high affinity (pM to nM). The binding of BPI to endotoxin promotes killing and clearance of gram-negative bacteria by preventing endotoxin from binding to the pro-inflammatory endotoxin receptor complex of cells consisting of mCD14, MD-2 and TLR4. Inhibits inflammation and apoptosis induced by. Most BPIs are intracellular, but plasma levels of BPI increase with neutrophil activation and degranulation. Stable BPIs include, but are not limited to, rBPI ₂₁ , rBPI ₂₃ , rBPI ₅₀ , rBPI (10-193) ala ¹³² and the N-terminal fragment of BPI having a molecular weight of about 20-25 kD. The preparation of BPI and the like is described in the art in publications such as US Pat. No. 6,268,345, US Pat. No. 6,599,880, US Pat. No. 5420019, US Pat. No. 5,980,897 and US Publication No. 2008/0031874. Yes.

  BPI and / or the like may be provided in combination with an antibiotic (at least one, one or more antibiotics). In some embodiments, the antibiotic is quinolone. In some embodiments, the quinolone is a fluoroquinolone, which typically has a fluorine atom attached to the central ring system at the 6- or 7-position. Examples of quinolone antibiotics administered in combination with BPI and / or the like include, but are not limited to, moxifloxacin, ciprofloxacin, levofloxacin, garenoxacin and delafloxacin.

  BPI and / or the like and antibiotics may be administered simultaneously or sequentially. When BPI and / or its congeners and antibiotics are administered simultaneously, they may be administered in the same formulation or in separate formulations, and are administered at substantially the same time. Administration of antibiotics and BPI and / or the like may also be sequential: only administering the two at a time sufficiently close to have the desired effect on bone marrow function. is necessary. In certain embodiments, the antibiotic is administered before BPI and / or the like or after administration of BPI and / or the like. The time separation between administrations of these compounds may be for a few minutes only, 5 hours, 12 hours, 24 hours, 48 hours or 96 hours, or longer.

  The compounds of the invention are administered in effective amounts. An effective amount is a dose sufficient to provide medically desirable results and can be determined by one skilled in the art using routine methods. In the treatment of radiation-induced tissue damage, an effective amount would be the amount necessary to inhibit (partially or completely) tissue damage caused by exposure to radiation. In some embodiments, an effective amount is an amount that provides an improvement in the condition being treated. In some embodiments, the effective amount may depend on the type and extent of radiation exposure and / or the use of one or more additional therapeutic agents. However, one of ordinary skill in the art will determine the appropriate dose and range of BPI / conservatives and antibiotics to use based on, for example, in vitro and / or in vivo testing and / or other knowledge of compound dosage. be able to. In some embodiments, the BPI and / or the like and the antibiotics described herein are non-cancer caused by radiation without materially interfering with the killing of cancerous tissues and cells. It should be understood that it can be administered in dosages that inhibit sexual tissue and cell injury.

  When administered to a subject, effective amounts of BPI / conservatives and antibiotics include, for example, severity of injury; individual patient parameters (including age, physical condition, size and weight), combination treatment, treatment The frequency of this will depend on the mode of administration. These factors are well known to those skilled in the art and can be addressed using only routine experimentation. In some embodiments, the maximum dose is used, that is, the maximum safe dose according to sound medical judgment.

  An effective amount is typically about 0.001 mg in administration of one or more doses, over a day or days or many days (depending on the mode of administration and the factors discussed above). / Kg to about 1000 mg / kg, about 0.01 mg / kg to about 750 mg / kg, about 0.1 mg / kg to about 500 mg / kg, about 1.0 mg / kg to about 250 mg / kg, about 10.0 mg / kg Can vary from ~ 150 mg / kg.

  The actual dosage level of BPI / conservatives and antibiotics can be varied to obtain an amount effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration. The dosage level chosen will depend on the activity of the particular compound, the route of administration, the severity of the radiation exposure, and the previous therapeutic history of the patient being treated. However, starting treatment with a dose of the compound at a lower level than required to achieve the desired therapeutic effort, and gradually increasing the dosage until the desired effect is achieved, Within the skill of the art.

  BPI and / or the like and antibiotics can be administered at any time after a subject has been diagnosed with a (partial or complete) decrease in bone marrow function. In some embodiments, the BPI and / or the like and the antibiotic are administered at any time after the subject has been diagnosed with decreased blood counts compared to expected normal levels. Can do. In some embodiments, the BPI and / or the like and the antibiotic are administered before, during or after exposure of the subject to a level of radiation that causes tissue damage. In some embodiments, the BPI and / or the like and the antibiotic are prior to radiation exposure but are sufficiently in time proximity to radiation exposure to inhibit radiation-induced tissue damage. Be administered. In some embodiments, BPI and / or the like and antibiotics are administered at any time up to 1 day prior to radiation exposure. In some embodiments, the BPI and / or the like and the antibiotic are administered 1-24 hours prior to radiation exposure. In some embodiments, BPI and / or the like and antibiotics are administered within 12 hours of radiation exposure. BPI and / or the like and antibiotics may also be administered during radiation exposure. In some embodiments, the BPI and / or the like and the antibiotic are radiation exposure after radiation exposure but sufficient to have the desired effect of protecting the tissue from radiation-induced tissue injury. Administered in close proximity to each other. In some embodiments, BPI and / or its congeners and antibiotics are administered at any time up to 3 days after exposure. In some embodiments, BPI and / or the like and antibiotics are administered 1 to 60 hours after radiation exposure. In some embodiments, the BPI and / or the like and the antibiotic are administered within 24 or 48 hours of radiation exposure. In some embodiments, the subject has cancer, and BPI and / or the like and antibiotics are at least 1 hour, at least 12 hours, at least 24 hours after radiation therapy, chemoradiotherapy or chemotherapy, Or at least 48 hours later, but after 72 hours after radiation therapy, chemoradiotherapy or chemotherapy.

  BPI and / or the like and antibiotics, and pharmaceutical compositions comprising BPI and / or the like and antibiotics are administered to the subject by any suitable route. For example, the composition can be orally (including sublingual), rectally, parenterally, from the cisterna, from the vagina, topically and transdermally (by powder, ointment or droplets). As), or from the mouth or from the nose. The term “parenteral” administration, as used herein, refers to modes of administration other than those through the gastrointestinal tract, which are intravenous, intramuscular, intraperitoneal, intrasternal, breast Intrammary, intraocular, retrobulbar, intrapulmonary, intrathecal, subcutaneous and intraarticular injection and infusion. Surgical implantation is also contemplated, including, for example, embedding the composition of the invention in a body, such as in the brain. In some embodiments, the composition can be administered systemically.

  The pharmaceutical compositions of the present invention for parenteral injection include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile injectable solutions just before use. Or aseptic powder for reconstitution into a dispersion. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol) and suitable mixtures thereof, vegetable oils (such as olive oil), and oleic acid Injectable organic esters such as ethyl. The proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

  These compositions may also contain preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of microbial activity can be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbate, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of injectable pharmaceutical preparations can be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

  In some cases, it is desirable to delay the absorption of the drug from subcutaneous or intramuscular injection in order to prolong the effect of the drug. This result can be achieved by the use of a liquid suspension of amorphous material with low water solubility. Delayed absorption of a parenterally administered drug is also achieved by dissolving or suspending the drug in an oil vehicle. Similarly, injectable depot preparations are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactic acid-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

  The present invention provides a method for oral administration of the pharmaceutical composition of the present invention. Oral solid formulations are generally described in Chapter 89 in Remington's Pharmaceutical Sciences, 18th edition, 1990 (Mack Publishing Co. Easton Pa. 18042). Solid formulations for oral administration include capsules, tablets, pills, powders, troches or lozenges, cachets, pellets, and granules. Liposomes or proteinoid encapsulation can also be used to formulate the compositions of the invention (eg, as proteinoid microspheres reported in US Pat. No. 4,925,673). Liposome encapsulation may include liposomes derivatized with a variety of polymers (eg, US Pat. No. 5,013,556).

  In such solid preparations, the active compounds are mixed with or chemically modified to contain at least one inert pharmaceutically acceptable excipient or carrier. Excipients or carriers can allow for compound uptake, overall stability of the compound, and / or increased circulation time of the compound in the body. Excipients and carriers include, for example: sodium citrate or dicalcium phosphate, and / or (a) packing such as starch, lactose, sucrose, glucose, cellulose, modified dextran, mannitol and silicic acid Or bulking agents, and inorganic salts such as calcium triphosphate, magnesium carbonate and sodium chloride, and FAST-FLO®, EMDEX®, STA-RX 1500®, EMCOMPRESS® and Commercially available diluents such as AVICEL®, (b) binders such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, gums (eg alginate, gum arabic), gelatin, polyvinylpyrrolidone and sucrose, (c ) Glycerol (D) disintegrants such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, sodium carbonate, starch containing disintegrants based on commercially available starch, EXPLOTAB® ), Sodium starch glycolate, AMBERLITE®, sodium carboxymethylcellulose, ultramylopectin, gelatin, orange peel, carboxymethylcellulose, natural sponge, bentonite, insoluble cation exchange resin, and agar, Karaya gum or tragacanth, etc. (E) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds and fatty acids including oleic acid, linoleic acid and linolenic acid, (g) wetting Agent For example, anionic detergent surfactants including cetyl alcohol and glycerol monostearate, sodium lauryl sulfate, sodium dioctylsulfosuccinate, and sodium dioctylsulfonate, cationic detergents such as benzalkonium chloride or benzethonium chloride, lauromacrogol 400, polyoxy40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbates 40, 60, 65 and 80, nonionic detergents including sucrose fatty acid esters, methylcellulose and carboxymethylcellulose, etc. (H) absorbents such as kaolin and bentonite clay, (i) talc, calcium stearate, magnesium stearate, solid polyethylene Glycols, sodium lauryl sulfate, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oil, wax, CARBOWAX® 4000, CARBOWAX® 6000, lubricants such as magnesium lauryl sulfate, and mixtures thereof; (j ) Glidants that improve the flow properties of the drug during formulation and assist in reconstitution during compression, including starch, talc, calcined silica and hydrated silicoaluminate. In the case of capsules, tablets and pills, the formulation may also include a buffering agent.

  The solid formulations of tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical art. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, liquid formulations contain certain inert diluents commonly used in the art (such as water and other solvents), solubilizers and emulsifiers (ethyl alcohol, isopropyl alcohol, ethyl carbonate, Ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, fats and oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydroflur Furyl alcohol, polyethylene glycol, sorbitan fatty acid esters, and the like, and mixtures thereof).

  Also contemplated herein is pulmonary delivery of the compounds of the invention. The compound is delivered to the mammalian lung during inhalation. Contemplated for use in the practice of the present invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powders All of these are well known to those skilled in the art, including inhalers. All such devices require the use of suitable formulations for the dispensing of the compounds of the invention. Typically, each formulation is specific to the type of device used and may involve the use of suitable spray materials in addition to diluents, adjuvants and / or carriers used in therapy.

  The invention is further illustrated by the following examples, which should in no way be construed as further limiting. The entire contents of all references cited throughout this application, including academic literature, issued patents, published patent applications and co-pending patent applications, are hereby incorporated by reference.

Examples Materials and methods Patient characteristics Patients undergoing myeloablative allogeneic HSCT (n = 48) at Boston Children's Hospital (CHB) or Brigham and Women's Hospital (BWH) in 2005-2009 In the study approved by the internal review committee, it was recruited in advance. All participants and / or legal guardians provided consent and / or consent as needed. Age ranged from 1 to 60 years. Bone marrow destruction regimens for hematological malignancies (n = 46) are chemoradiotherapy with 1400 cGy (n = 38) or 1375 cGy (n = 1) TBI, or> 14 mg / kg oral or IV equivalent Combination chemotherapy with an amount of busulfan (n = 7). Bone marrow destruction for aplastic anemia was cyclophosphamide 200 mg / kg (6 gm / M ² ) plus ATG (n = 2). Sixteen patients received BM and 32 received PB stem cells. Supportive care followed institutional established procedures (48, 49). Prophylactic oral non-absorbable antibiotics were administered: bacitracin and either polymyxin (BWH) or vancomycin (CHB). Hematology and culture were performed according to clinical laboratory defined procedures. Sixteen patients had bacteremia due to either gram positive (n = 15) or gram negative (n = 1) organisms. Body temperature was recorded using the maximum value within ± 1 day of sample acquisition. Endotoxin activity assay (EAA) measurements were added when EAA was available.

Blood collection and plasma preparation PB samples are drawn into K2-EDTA or sodium heparin Vacutainers ™ (Becton-Dickinson (BD), Franklin Lakes, NJ) (baseline, B), then HSCT day (D0) , And weekly ± 1 day pretreatment. PB was centrifuged at 1200 g for 5 minutes at 4 ° C., collected, and stored in aliquots at −80 ° C. in pyrogen-free tubes.

Human BPI ELISA
BPI was measured by ELISA (HyCult, Uden, The Netherlands) according to manufacturer's instructions.
Endotoxin measurement in human PB Endotoxin was measured by EAA according to the manufacturer's instructions (Spectral Diagnostics, Toronto, Canada) (27).

Human IL-6 ELISA
IL-6 was performed by flow cytometry (MoFlo, DakoCytomation, Glostrup, Denmark) using antibody-coated fluorescent beads (Cytometric Bead Array BD Flex Sets, BD BioSciences, San Jose, CA) and Summit v4.0 software (DakoCytomation). Measured.
Measurement of mCD14 and TLR4 Monocyte surface expression of CD14 and TLR4 was measured by antigen-specific or isotype control monoclonal antibodies (eBioSciences, San Diego, CA) as previously described (50).

In vivo radiation relaxation studies with rBPI ₂₁ and enrofloxacin Male BALB / c mice (stock # 028, Charles River, Wilmington, Mass.) were acclimated prior to irradiation at 12 weeks of age. The study was conducted according to policies and protocols approved by ACUC of Dana-Farber Cancer Institute. Mice were placed in a Rad Disk ™ rodent microisolation irradiation cage (Braintree Scientific, Braintree, MA) and 1% with a Gammacell® 40 Exactor (Best Theratronics, Ottawa, Ontario) cesium source irradiator. A dose of 7 Gy was administered. Twenty-four hours later, mice were left untreated (7 Gy) or received one or more of the following treatments for 30 days: 1) rBPI ₂₁ (XOMA (US) LLC, Berkeley, CA) 250 μl per injection of 2 mg / ml stock was made up in formulation buffer and administered SC twice a day at 6-8 hour intervals (rBPI ₂₁ / mouse was ~ 42 mg 2) 0.33 g / L citric acid, 1.01 g / L sodium citrate, 8.76 g / L sodium chloride, 2.0 g / L Poloxamer P188, and 2 250 μl of rBPI ₂₁ formulation buffer (designated VEH) consisting of 0.0 g / L polysorbate 80 (all Sigma, St. Louis, MO) was dissolved in water for injection and the pH was adjusted to 5.0; Sterilized by filtration; 3) Baytril ®) (enrofloxacin, MedVets, Sandy, UT) for the first 5-7 days by gavage via a 25G dietary needle (Cadence Science, Cranston, RI) at 10 mg / kg / day The mice were then dosed in ad lib water bottles until the end of the study or until death. All mice were observed at least twice daily. Drowned mice were euthanized by CO ₂ asphyxiation. At planned time points, mice were euthanized with an overdose of isoflurane anesthesia (IsoFlo® Abbott Labs, Abbott Park, IL).

Blood and tissue preparations CBCs were performed in Heemavet 950 FS hematology analyzers (Drew Scientific, Waterbury, Conn.) Using cardiac blood anticoagulated with EDTA (Becton-Dickinson, Franklin Lakes, NJ). Plasma was obtained by mixing the blood with pyrogen-free heparin (APP Pharmaceutical, Schaumburg, IL) in a pyrogen-free eppendorf tube (USA Scientific) and centrifuging at 14,000 rpm for 10 minutes. Single use aliquots were stored at -80 °. In some studies, the femur and tibia from one limb per animal were dissected and fixed and treated (coronary) in 10% neutral buffered formalin (Fisher Scientific, Pittsburg, PA). Section preparation and hematoxylin-eosin (H & E) staining (including Specialized Histopathology Services-Longwood, Boston, MA). Flush cells from bone with cold RPMI 1640 medium supplemented with 10% FBS (JRH Biosciences, Lenexa, KS), L-glutamine, HEPES, penicillin / streptomycin and gentamicin (all from Invitrogen, Carlsbad, CA). The contralateral femur and tibia were used for BM MNC enumeration and flow cytometry. Red blood cells were lysed with hypotonic lysis buffer (Sigma). BM MNCs were enumerate by trypan blue staining; viability was typically> 90-95%.

Citrulline determination Samples were analyzed by MassTrak Amino Acid Analysis (AAA) system (Waters, Milford MA USA) using AccQTag ™ derivatization and UV / visible detection.

Histopathological evaluation A qualified hematopathologist (JK), using an Olympus BX51 microscope and an Olympus DP71 camera, in a H & E-stained paraffin-fixed section demineralized and formalin-fixed, confirms femoral BM cell integrity. Evaluated with DP Capture software. For each animal, 2 slides were scored at 2 fields / slide for the percentage of BM space lined by hematopoietic cells. A qualified pathologist (J-AV) counted apoptotic bodies / 400 × field in three replicate samples of H & E stained paraffin-fixed coronal sections. Samples from normal mice identified identities, but all others were deidentify and presented for analysis in a random order.

Endotoxin measurement in mouse plasma Endotoxin was measured using the Limulus Amebocyte Lysate (LAL) assay according to the manufacturer's instructions (Charles River, Boston, MA) and as previously described (51). .

BM FACS analysis BM cells were preincubated with 2% rat anti-mouse CD16 / CD32 and 1% normal rat serum for Fc blocking, followed by hematopoietic lineage markers (CD3ε, CD45 / B220, CD11b, Ly-6G / Cells with Ly-6C, TER 119) were added to a cocktail of APC conjugated lineage specific antibodies, or equal concentrations of APC conjugated isotype control immunoglobulin, 1:20 dilution of PE-rat anti-mouse Sca 1 (clone D7) and PerCP. -Stained with Cy5.5-rat anti-mouse c-Kit (clone 2B8) (all from BD). Cells were stained for 25 minutes at 4 ° C., washed twice with cold DPBS, and resuspended in 0.4% paraformaldehyde. 100,000 events were acquired on a FACScalibur ™ flow cytometer (BD) and analyzed with FlowJo v. 7.0.5 (Treestar) software. Cells negative for lineage markers were evaluated for the percentage of lin ^- Sca-1 ^- c-kit ⁺ (LK) and lin ^- Sca-1 ⁺ c-kit ^{+ (} LSK) in BM (FIG. 10, gate strategy). . Data from normal mice was consistent with published reports for naive BALB / c mice (52).

Statistics For human HSCT studies, samples with undetectable analytes were assigned a value at half the lower limit of detection. Data was analyzed after logarithmic transformation for ANC, PLT, BPI, TLR4 and IL-6. This is because it results in a more regular close distribution. For these data, error bars representing the geometric mean of logarithmic values plus one standard error of the mean (SEM) were then converted back to the original units and plotted on the log axis. When comparing values for the same patient at different time points, the Wilcoxon signed rank test for matched pairs was used, using the values compared to the baseline. Comparisons between subjects with or without fever were evaluated using the Mann-Whitney test. When assessing correlations between different parameters, intra-subject correlations were calculated using Spearman correlation coefficients and data from multiple time points. The calculated counts were averaged across different subjects and tested for significance by a signed rank test. Unless otherwise noted, all p values were bilateral. Statistical significance and graphic output were generated using Prism v. 4.0a (GraphPad Software; San Diego, CA) and SAS v. 9.1 (SAS Institute, Cary, NC). Statistical analysis for mouse experiments was performed with Graph Pad Prism version 5. Survival curves were compared using the Mantel-Cox log rank. Citrulline data (where data was analyzed by comparison with 100% theoretical mean by 1 sample t-test) and hematology analysis (Table 1) (where unpaired t-test was performed) A two-tailed t-test (Mann-Whitney) was performed throughout. Unpaired t tests do not assume equivalent variances. In all experiments, reject the null hypothesis with a P value <0.05. When shown in the figure, * p <0.05, ** p <0.01, *** p <0.001.

  In the shaded portion, the median shows a value corresponding to the normal range of the 0 Gy control with the same age. Statistically significant for 0 Gy; statistically significant for 7 Gy; ■ statistically significant for VEH / ENR; ▼ statistically significant for ENR. All p <0.05.

Results Human bone marrow destruction HSCT is associated with evidence of early neutropenia, endotoxemia, BPI deficiency and host response to endotoxins.
We tested plasma levels of endotoxin and BPI in patients who received bone marrow destruction treatment for HSCT. Of 48 patients, 39 received chemoradiotherapy including 1375 (n = 1) or 1400 (n = 38) cGy TBI, while 9 received only chemotherapy combined with destruction . As expected, allogeneic HSC infusion following myeloablative treatment resulted in a decrease and recovery of PB count (FIG. 1A). By the completion of the bone marrow destruction procedure (D0), endotoxemia was easily detectable (FIG. 1B). At the same time, plasma BPI concentrations declined rapidly (median D7 decreased 71-fold, interquartile range decreased 9-193 fold; FIG. 1B), which was measured by absolute neutrophil count and Correlated (ANC; Spearman r = 0.66; p <0.001). At the ANC bottom point (D7), plasma BPI is undetectable in 37/48 patients (77%) (<100 pg / mL), and 80% (27) of patients evaluated by endotoxin activity assay It was endotoxemia.

  The TLR endotoxin receptor TLR4 and mCD14 components on peripheral blood (PB) monocytes show increased surface expression and phenomenon, respectively, consistent with exposure of early BP monocyte cells to bioactive endotoxin (28 29) (FIG. 1C). The well-described downstream sequelae of TLR4, the subsequent rise in IL-6 and fever, were maximal at the BPI nadir (D7, FIG. 1D). Intra-patient changes in IL-6 concentrations were positively correlated with EAA (Spearman: 0.48, p = .01), and higher levels of IL-6 were inversely correlated with BPI levels (Spearman:-. 30, p <.0001). Fever and BPI levels do not show any association at D7, probably because nearly 80% of patients had undetectable BPI. However, at D14, patients with fever had lower BPI levels than those with no fever (median: undetectable vs. 3475 pg / mL, p = .01). Importantly, lower plasma BPI concentrations at D0 just prior to HSC infusion are associated with longer time neutrophil engraftment (p = .03, Spearman r = −. 32) and time to platelet recovery Associated with the trend of longer (Spearman r = −. 26, p = .08).

  These results suggest that a deficiency of BPI coupled with endotoxemia may contribute to endotoxin-related toxicity following bone marrow destruction, and that supplementation with BPI may attenuate these toxicities. suggest. Although administration of HSC allows survival after bone marrow destruction, support for HSC is not possible after unintentional radiation exposure. Since relaxation of radiation without HSC cannot be addressed experimentally in humans, we tested this hypothesis using a mouse model.

Characterization of the toxicity of 7 Gy single-irradiated TBI in BALB / c mice In order to model the potentially lethal radiation exposure, we have determined that BM hypoplasia, GI toxicity and The dose of single-irradiated TBI associated with a high rate of early death was defined. A single dose of _{7 Gy} was associated with 95-100% mortality (LD _95/30 ) up to 30 days in 12-week-old BALB / c (FIG. 2A). The lethality of 7 Gy exposure was reproducibly observed in each of the subsequent relaxation experiments: only 5/90 7 Gy irradiated mice (5.5%) survived to D30, with survival rates in separate experiments. The median ranged from 12-15 days. Small intestinal epithelial apoptosis assessed by histopathology after 7 Gy TBI was maximal in D3 and paralleled a decrease in plasma citrulline levels directly proportional to functional GI enterocyte mass (30) (FIG. 2B). ). Both GI mucosal findings improved by D6-9. Endotoxemia was also detectable by D3 and persisted until a higher spike just before death (FIG. 7). By D3, BM was aplastic (Fig. 2C), and the content of BM monocytes (MNC) decreased simultaneously by nearly two logarithms, which is the same as hematopoietic stem (LSK, Lin ^- Sca-1 ⁺ c-Kit ⁺ ) and precursor (LK, Lin ⁻ Sca-1 ⁻ c-Kit ⁺ ) cell decay (FIGS. 2D, E, F).

  The degree of mucosal injury, inflammation and toxicity experienced during human HSCT has been linked to the intensity of bone marrow destruction (31). To confirm that the model was properly myeloablative, we compared the effects of 7 and 6.5 Gy on hematopoiesis. Tissue hypoplasia was the same at D3 regardless of TBI dose, but mice had significantly more BM MNC, LK and LSK3 than 7 Gy after 10 and 15 days of 6.5 Gy (FIG. 2D). , E, F). In the 7Gy cohort, no significant recovery was observed by D15. Subsequent relaxation experiments were performed after exposure to 7Gy.

rBPI ₂₁ and enrofloxacin (ENR) administration significantly reduce TBI-related mortality
and rBPI _21, in combination with oral ENR is fluoroquinolone antibiotic similar to ciprofloxacin (rBPI ₂₁ / ENR), started 24 hours after 7Gy the TBI dose of LD _95/30, until D30 This resulted in a statistically significant improvement in mouse survival at D30 (FIG. 3). In a composite analysis of two replicate experiments (accumulating n = 50 mice / arm), the viability of the rBPI ₂₁ / ENR group is that of VEH / ENR (VEH represents the formulation buffer for rBPI ₂₁ ) , As well as ENR or 7Gy alone groups (<.0001, 0.03 and <.0001, respectively, in the pair-wise Mantel-Cox log rank). Of the 36 rBPI ₂₁ / ENR-treated mice at risk, only 2 deaths occurred after 2 weeks, while the loss in the other groups during this interval occurred among the animals at risk Of 38 to 79%. The survival rate of D30 after 7 Gy was not improved by either rBPI ₂₁ (1/30 survivor) or its VEH (1/30 survivor) alone. Therefore, rBPI ₂₁ monotherapy was not explored as a palliative strategy at the TBI dose of LD _95/30 .

When administered by IV bolus or subcutaneous (SC) injection, rBPI ₂₁ has a half-life of 3 hours in mice. Since an optimal sustained or 96-hour IV or SC injection regimen is not possible, we use twice daily SC administration and all treatments begin 24 hours after TBI and continue until D30 Chose to do. As illustrated in FIG. 3, VEH / ENR, the control for the rBPI ₂₁ / ENR regimen, was associated with a worse 30-day survival rate than oral ENR alone (two combined n = 50 / arm 2 times). According to the pair-wise Mantel-Cox log rank for replication experiments, p = 0.0002), this suggested that repeated handling and local skin trauma associated with SC administration was associated with significant toxicity. Local skin injury is readily observed in irradiated mice injected repeatedly with rBPI ₂₁ or VEH compared to irradiated mice treated with ENR alone (FIG. 8).

A shortened schedule for stopping the injection after 14 days (represented as rBPI ₂₁ (14) and VEH (14) in FIG. 3B) was investigated. The ENR schedule was not changed in a mass disaster setting because oral antibiotic treatment can be more easily deployed. rBPI ₂₁ (14) / ENR had the same survival benefit as the longer schedule (FIG. 3B). Six irradiated mice administered rBPI ₂₁ (14) / ENR were followed to D30 and 5 of the 6 mice continued to survive at D131 and appeared healthy.

To characterize the effect of rBPI ₂₁ / ENR on hematopoiesis that alleviates hematopoietic toxicity after TBI, we factored in the BM MNC recovered from the washed-out BM cavity (FIG. 4). At D10, all irradiated groups showed less BM MNC than unirradiated controls matched for age regardless of treatment (p <0.0001). However, BM MNC content was significantly higher in rBPI ₂₁ / ENR-treated mice than in mice that received 7 Gy alone or with ENR or VEH / ENR (p = .0003, 0.001 and <. 0001). This same pattern was repeated at D15 and D19 because rBPI ₂₁ / ENR treated mice consistently had a statistically significantly higher BM MNC content than the other groups (FIG. 4). rBPI ₂₁ / ENR treatment was associated with a consistently higher BM MNC content than observed after 7 Gy alone.

This rapid increase in BM MNC was reflected in the cellularity of BM assessed by pathological tissue at D19 (FIG. 4). Irradiated mice treated with rBPI ₂₁ / ENR show well recovered BM cell solidity in the range of 80-90%, whereas mice treated with 7Gy alone, ENR, and VEH / ENR, respectively, Estimated to be 20, <5, and 10-50%. Hematopoietic hematopoiesis was observed in all mice with sufficient cellularity, and a subset of mice, particularly those administered rBPI ₂₁ / ENR, showed myelocyte dominance and megakaryocyte increase ( FIG. 9). By D30, all surviving mice showed improved cellularity, as previously described in mouse TBI survivors (32). However, rBPI ₂₁ / ENR treated mice showed stronger cell integrity and had significantly more BM MNC than ENR or VEH / ENR (FIG. 5). Restored cellularity was also observed in a more limited pool of 7 Gy survivors at D30.

The number of LSK and LK remained lower at each time point than matched unirradiated controls, but administration of rBPI ₂₁ / ENR resulted in the number of LSK and LK BM cells in the first week after TBI. (Fig. 6). The absolute numbers of both LSK and LK per hind limb in rBPI ₂₁ / ENR mice were significantly higher in D10 and D19 than in 7Gy alone, ENR or VEH / ENR treated mice. Other treatments were not related to differences from the untreated irradiated group. At D30, there was no difference between surviving mice between treatment groups or between treatment and normal controls.

Changes in BM correlated with changes in peripheral blood counts. The effect of 7 Gy on PB count could be observed in virtually all measured hematological parameters (Table 1). rBPI ₂₁ / ENR treatment was associated with greater recovery of leukocytes (WBC), neutrophils, monocytes and platelet counts than 7 Gy alone, ENR or VEH / ENR treatment. In contrast to the other groups, the median WBC, neutrophil and monocyte levels of rBPI ₂₁ / ENR treated mice were within normal limits. The median hemoglobin was also higher in rBPI ₂₁ / ENR treated mice, but this difference did not reach statistical significance. The WBC and neutrophils of rBPI ₂₁ / ENR-treated mice remain significantly higher than ENR-treated animals at D30, and 7Gy alone or VEH / ENR mice present at this time are significant with the other groups Too little for comparison. Equivalent hematopoietic toxicity alleviation was observed with the shorter rBPI ₂₁ (14) / ENR schedule (FIGS. 12 and 13).

rBPI ₂₁ administration significantly increases inflammation-related chemokines
rBPI ₂₁ treatment consists of granulocyte colony stimulating factor (G-CSF) (FIG. 14), mouse keratinocyte chemotactic factor (mouse KC; human homologs include Gro-alpha, IL-8) (FIG. 15), And inflammation-related chemokines such as monocyte chemotactic protein-1 (MCP-1, aka chemokine (CC motif) ligand 2 or CCL2) are significantly increased (FIG. 16). BPI stimulation of G-CSF and mouse KC in plasma is enhanced by prior radiation but is not isolated thereto. While not intending to be bound by theory, this represents one mechanism by which the hematopoietic effect is realized. The plasma levels achieved after irradiation and rBPI administration are consistent with infusion of IV pharmacological amounts of recombinant G-CSF. The data shows a highly significant increase (1-3 log increase) in G-CSF in irradiated mice administered rBPI alone or in combination with enrofloxacin twice daily (BID) in SC. Thus, the increase in GCSF is a function of rBPI and does not depend on the combination of BPI and ENR. Less significantly, but a statistically significant increase in G-CSF was also observed in unirradiated mice that received either a single rBPI injection or a twice daily (BID) SC injection. It was done.

An observational cohort study in patients undergoing HSCT was conducted to identify molecular and cellular changes that could be involved in the toxicity of myeloablative irradiation. The inventors have shown that neutropenia that always follows bone marrow destruction treatment is a protein with potent endotoxin neutralizing activity induced by neutrophils (25, 26) and rapid depletion of plasma BPI; We observed that it was related to endotoxemia simultaneously in time. These changes paralleled changes in cells (mCD14, TLR4 surface levels), plasma (IL-6) and physiological (fever) consistent with increased systemic endotoxin activity (24, 25). We also observed that lower plasma BPI concentrations at the time of HSC infusion (D0) correlated with slower myelocyte engraftment, indicating that endotoxin was at the time of infusion and Suggests that it will exert some negative or direct impact on HSC over the subsequent period. The ability of exogenous BPI supplementation to alleviate radiation toxicity in humans exposed to TBI doses resulting in mucosal injury, endotoxemia and long-term BM formation was then investigated. Using a bone marrow disruption TBI model with a single _dose of LD _95/30 in BALB / c mice, we found that the combination of rBPI ₂₁ and ENR initiated 24 hours after radiation exposure was two-thirds. It was also shown to be related to the survival of more animals (p <0.0001). We selected rBPI ₂₁ and fluoroquinolone antibiotics as a readily viable strategy; both agents are healthy and sick humans, including those with multi-organ susceptibility Have a biological activity and a highly favorable safety profile (33-43). rBPI ₂₁ alone did not improve survival, whereas ENR alone provided some survival benefit. The mitigating effects of fluoroquinolone alone are variable, potentially related to differences including animal models and treatment design (15, 16). In this study, the survival benefit of ENR treatment was significantly lower than that of rBPI ₂₁ / ENR, regardless of repeated injury of the injection regimen. In addition, irradiated animals treated with VEH / ENR or ENR were characterized by delayed recovery of all hematopoietic parameters tested. Only rBPI ₂₁ / ENR was consistently associated with both improved survival and faster and more complete hematopoietic recovery. These may be related to the findings suggested by 97% survival of rBPI ₂₁ / ENR treated animals after near-normal BM cell integrity and PB count reconstitution recorded at D19.

The contribution of hematopoietic syndrome to ARS morbidity and mortality in humans (1, 2, 4, 44-46) highlights the involvement of the observed effect of rBPI ₂₁ / ENR on hematopoietic development. Allogeneic HSCT alleviates BM failure caused by bone marrow destruction (5), but it is unlikely that resource-intensive HSCT can be transplanted quickly and successfully during mass radiation exposure (44-46). Multiple agents (47) including fluoroquinolone (16) and the TLR agonists flagellin (19, 21) and endotoxin (17) provide some radioprotection when administered prior to TBI in animal models. In contrast, few agents show efficacy (ie, radiation relaxation) when administered after radiation, and efficacy has generally depended on administration within minutes to hours after radiation. Unfortunately, such rapid deployment of mitigation strategies is unlikely, making a strategy that may be delayed for 24 hours or longer highly desirable.

There are no established radiation dosimetry techniques that can accurately triage exposed individuals and determine the individuals most likely to benefit from palliative treatment, reducing the efficacy and toxicity of radiation mitigants. There is also no human therapeutic application of TBI without the support of HSC to study. These limiting factors underscore the importance of choosing strategies that are unlikely to cause toxicity in either the least affected or severely ill populations. The components of the strategy studied here satisfy this criterion. The human equivalent of ENR, a fluoroquinolone veterinary drug, is ciprofloxacin, which was approved by the FDA in 1987. Fluoroquinolones have excellent oral bioavailability, are well tolerated, and have been used extensively and safely after myeloablative chemoradiotherapy (42, 43). rBPI ₂₁ is available in a soluble form and has demonstrated stability when stored at 2-8 ° C. which facilitates stockpile. It can be administered SC, IV and IP and has shown efficacy in intranasal form in animals. In addition to efficacy in animal models of pure endotoxemia and gram-negative bacteremia, rBPI ₂₁ suppresses signs and symptoms of endotoxemia in humans and reduces related cytokine dysregulation and coagulation damage Or it can be removed (38, 39). In Phase I-III trials enrolling more than 1100 normal volunteers and severely ill patients, including children and subjects with meningococcal bacteremia or experiencing major surgical procedures No significant toxicity has been observed (33-41). In a pilot experiment (n = 4), we also administered rBPI ₂₁ to patients undergoing TBI as part of myeloablative HSCT but without any toxicity due to it (FIG. 11). ). Taken together, these data suggest that rBPI ₂₁ / ENR can be safely administered to individuals who have been exposed to less well-documented levels of radiation.

Increasing global concern about radiation injury as a result of natural disasters, nuclear wars or terrorism, or as a detrimental result of deliberate medical exposure has made us effective in supplementing BPI. Led to a survey on whether it could be translated into a new radiation mitigation strategy. Our data show that the combination of rBPI ₂₁ and fluoroquinolone antibiotics initiated as much as 24 hours after radiation exposure, which can be lethal, improves survival and the range of supportive care required and It suggests having both the ability to limit the duration. In view of the relatively low sensitivity compared to humans in mice against endotoxins, suboptimal doses, and the repetitive stress and inflammatory response of SC injection in this model, our results indicate that this combination Potential benefits may be underestimated. The observed efficacy of treatments initiated 24 hours after TBI is hardly shared by other approaches and is emphasized in this advantage. The human safety record of rBPI ₂₁ and fluoroquinolone provides a platform for rapid adoption, which is particularly persuasive given the essential overtreatment resulting from the current limiting factors of radiation dosimetry. Yes, the potential for unexpected side effects can be reduced. In addition to neutralizing endotoxins, rBPI ₂₁ exerts antibacterial activity, which, in addition to the antibiotic activity of fluoroquinolone, potentially reduces further multidrug combinations and many reasons for infection The appearance of resistant species in irradiated individuals with can be minimized. This report provides the basis for exploring the mechanisms by which rBPI ₂₁ affects radiotoxicity. Optimization of the prescription, dosing regimen and length of treatment for rBPI ₂₁ or similar agents is equally desirable, but consideration of rBPI ₂₁ approval for this indication and subsequent combination palliative treatment in the event of a radiation disaster Stockpiling for is considered legitimate.

References

Claims (37)

A method for alleviating tissue injury resulting from exposure to radiation in a subject comprising:
Including administering to a subject in need thereof a bactericidal / permeability enhancing protein (BPI) and / or the like in an effective amount to alleviate tissue injury caused by the subject's radiation exposure. , Said method.   The method of claim 1, wherein the tissue injury comprises hematopoietic toxicity.   The method according to any one of claims 1-2, wherein the radiation exposure results from accidental exposure to radiation, chemoradiotherapy or radiation therapy. BPI congeners _is selected from the group consisting of N-terminal fragment of BPI having an approximate molecular weight of _{rBPI 21, rBPI 23, rBPI 50} , rBPI (10-193) ala 132 and about 20kD~ about 25 kD, claim 1 The method as described in any one of -4.   5. The method according to any one of claims 1 to 4, further comprising administering an antibiotic.   The method according to claim 5, wherein the antibiotic is a quinolone antibiotic.   7. The method of claim 6, wherein the quinolone antibiotic is selected from the group consisting of moxifloxacin, ciprofloxacin, levofloxacin, garenoxacin and delafloxacin.   8. The method of any one of claims 1-7, wherein BPI and / or the like are administered from 1 day before to 2 days after exposure of the subject to radiation.   9. The method of claim 8, wherein BPI and / or the like are administered within 48 hours after radiation exposure.   10. The method according to any one of claims 1 to 9, wherein BPI and / or the like are administered orally, intravenously or subcutaneously. A method for mitigating hematopoietic toxicity resulting from exposure to radiation in a subject comprising:
Administering the bactericidal / permeability enhancing protein (BPI) and / or the like to a subject in need thereof in an effective amount to alleviate the hematopoietic toxicity of the subject.   12. The method of claim 11, wherein the hematopoietic toxicity results from accidental exposure to radiation, chemoradiotherapy or radiation therapy. BPI congeners _{are, rBPI 21, rBPI 23, rBPI} 50, rBPI (10-193) ala selected from ¹³² the group consisting of N-terminal fragment of BPI having a molecular weight of about 20～25KD, according to claim 11 to 12 The method according to any one of the above.   14. The method according to any one of claims 11 to 13, further comprising administering an antibiotic.   The method according to claim 14, wherein the antibiotic is a quinolone antibiotic.   16. The method of claim 15, wherein the quinolone antibiotic is selected from the group consisting of moxifloxacin, ciprofloxacin, levofloxacin, garenoxacin and delafloxacin.   17. The method of any one of claims 11 to 16, wherein BPI and / or the like are administered from 1 day before to 2 days after exposure of the subject to radiation.   18. The method of claim 17, wherein BPI and / or the like are administered after radiation exposure but within 48 hours of radiation exposure.   18. A method according to any one of claims 11 to 17, wherein BPI and / or the like are administered orally, intravenously or subcutaneously. A method for bone marrow recovery in a subject comprising:
The method comprising administering to a subject in need thereof a bactericidal / permeability enhancing protein (BPI) and / or the like in an effective amount for bone marrow recovery in the subject.   21. The method of claim 20, wherein the subject has one or more hematopoietic cell type or lineage deficiencies.   Subject is lymphopenia, myelocytopenia, leukopenia, neutropenia, erythropenia, megakarycytopenia, platelet deficiency, monocyte deficiency, lymphocyte deficiency, erythrocyte deficiency, 22. A method according to any one of claims 20 to 21 having a hematopoietic deficiency including neutrophil deficiency, T cell deficiency, granulocyte deficiency, and / or dendritic cell deficiency.   23. The method of claim 22, wherein the deficiency of one or more hematopoietic cell types or lineages results from radiation exposure, chemoradiotherapy, radiation therapy, disease, toxins or drugs or biologically mediated therapy. BPI congeners _{are, rBPI 21, rBPI 23, rBPI} 50, rBPI (10-193) ala selected from ¹³² the group consisting of N-terminal fragment of BPI having a molecular weight of about 20～25KD, according to claim 20 to 23 The method according to any one of the above.   25. The method according to any one of claims 20 to 24, further comprising administering an antibiotic.   26. The method of claim 25, wherein the antibiotic is a quinolone antibiotic.   27. The method of claim 26, wherein the quinolone antibiotic is selected from the group consisting of moxifloxacin, ciprofloxacin, levofloxacin, garenoxacin and delafloxacin.   28. A method according to any one of claims 20 to 27, wherein BPI and / or the like are administered orally, intravenously or subcutaneously. A method for stimulating hematopoiesis in a subject comprising:
Said method comprising administering to a subject in need thereof bactericidal / permeability enhancing protein (BPI) and / or the like in an effective amount to stimulate hematopoiesis in said subject.   30. The method of claim 29, wherein the subject has one or more hematopoietic cell type or lineage deficiencies.   Subject is lymphopenia, myelocytopenia, leukopenia, neutropenia, erythropenia, megakarycytopenia, platelet deficiency, monocyte deficiency, lymphocyte deficiency, erythrocyte deficiency, 31. The method of any one of claims 29-30, having a hematopoietic defect comprising neutrophil deficiency, T cell deficiency, granulocyte deficiency, and / or dendritic cell deficiency.   32. The method of claim 31, wherein the deficiency of one or more hematopoietic cell types or lineages results from radiation exposure, chemoradiotherapy, radiation therapy, disease, toxins or drugs or biologically mediated therapy. BPI congeners _{are, rBPI 21, rBPI 23, rBPI} 50, rBPI (10-193) ala selected from ¹³² the group consisting of N-terminal fragment of BPI having a molecular weight of about 20～25KD, according to claim 29 to 32 The method according to any one of the above.   34. The method according to any one of claims 29 to 33, further comprising administering an antibiotic.   35. The method of claim 34, wherein the antibiotic is a quinolone antibiotic.   36. The method of claim 35, wherein the quinolone antibiotic is selected from the group consisting of moxifloxacin, ciprofloxacin, levofloxacin, garenoxacin and delafloxacin.   37. The method of any one of claims 29 to 36, wherein BPI and / or the like are administered orally, intravenously, or subcutaneously.